Cyclic Ion Mobility-Collision Activation Experiments Elucidate Protein Behavior in the Gas Phase

Area of Science:

• Biophysical Chemistry
• Structural Biology
• Analytical Chemistry

Background:

• Ion mobility coupled to mass spectrometry (IM-MS) is a powerful tool for gas-phase protein structure and dynamics.
• Protein unfolding can be induced by increasing ion energy in the IM cell, yielding insights into proteoform energetics.
• Cyclic IM-mass spectrometry (cIM-MS) enables multiple, consecutive tandem IM experiments (IMⁿ).

Purpose of the Study:

• To describe a tandem IM technique for detailed protein unfolding pathways and disordered protein dynamics.
• To apply this IMⁿ-MS method to model proteins like cytochrome C and human islet amyloid polypeptide (hIAPP).

Main Methods:

• Utilized a tandem IM technique involving multiple rounds of IM separation and collision activation (CA): IM-CA-IM and CA-IM-CA-IM.
• Applied the IMⁿ-MS method to cytochrome C and dimeric hIAPP.
• Analyzed unfolding events and structural interconversions.

Main Results:

• Observed multiple unfolding events for cytochrome C, consistent with previous IM-MS studies.
• Detected interconversion between compact and extended structures for both cytochrome C and hIAPP using IMⁿ-MS.
• hIAPP data indicated conformational interconversion prior to dissociation, suggesting low energy barriers.

Conclusions:

• Tandem IM techniques (IMⁿ-MS) provide detailed insights into protein unfolding pathways and dynamics.
• The method is effective for studying complex proteins and peptides, including amyloidogenic ones.
• Conformational flexibility and low energy barriers play a role in the dynamics of proteins like hIAPP.